Fig 2: Model for HG and inflammation-induced Acsl1 expression by CHREBP and NF-kappa B in macrophages.A) We propose that in high glucose, CHREBP is depressed, and active nuclear CHREBP promotes the expression of Acsl1. B) Under inflammatory conditions that activate the NF-kappa B via dismissal of the inhibitory protein I kappa B (IkB) that prevents NF-kappa B nuclear transport, Acsl1 transcription is induced. C) Acsl1 expression can be further increased by high glucose and inflammatory stimuli via CHREBP and NF-kappa B.

Fig 3: CHREBP contributes to transcriptional upregulation of Acsl1 in HG.A) CHREBP expression plasmid or vector only (VO) were co-transfected with Acsl1-GLuc reporter in HEK293 cells cultured under HG conditions. Luciferase assay was performed 48 hours post-transfection and shown as relative luciferase units (RLU). Expression of transfected Myc-tagged CHREBP is shown by western blot using a c-Myc antibody, with tubulin as a loading control. B) Wild type or Chrebp-/- BMDMs were differentiated under HG conditions. Acsl1 mRNA absolute copy number was quantified by qPCR. The data presented are means ± standard error (n = 3); the p-value was calculated using student’s t-test. (*p < 0.05).

Fig 4: Acsl1 expression is upregulated in macrophages by HG.A) BMDMs were differentiated under normal glucose (NG) and high glucose (HG), and Acsl1 mRNA absolute copy number was determined by quantitative real-time PCR (qPCR) using a standard curve generated with known quantities of mouse Acsl1 cDNA. B) Western blot of total cell lysates from BMDMs cultured in NG and HG using antibodies against ACSL1 and ß-actin as (loading control). C) The ratio of ACSL1/ß-actin band intensities was quantified using Image studio 5.2 (n = 3). D) Nascent Acsl1 RNA expression in BMDMs under NG and HG conditions was determined by qPCR. Nascent Acsl1 expression was quantified relative to cyclophilin A and shown as fold change. The data presented are the means ± standard error (n = 3); the p-value was calculated using the student’s t-test. (*p < 0.05).

Fig 5: ACSL1 protein abundance and membrane localization increase under inflammatory conditions.BMDMs were differentiated under NG conditions. A) Total cell lysates were prepared with (+) and without (-) LPS treatment (10 ng/ml for 24 hours). ACSL1 protein expression was determined by western blot with an anti-ACSL1 antibody. An anti-alpha tubulin antibody was used as a loading control. B) Bands were quantified using the Image studio 5.2. C) Cytoplasmic and membrane proteins were isolated to determine the localization of ACSL1 protein by western blotting as a function of LPS treatment. Alpha-tubulin (cytoplasmic protein) and Na+/K+ ATPase (membrane-associated protein) were used to confirm the fidelity of fractionation. D) Images were quantitated as in B with cytoplasmic ACSL1 protein normalized to tubulin and membrane ACSL1 protein normalized to Na+/K+ ATPase. The data presented are means ± standard error (n = 3); the p-value was calculated using student’s t-test. (*p < 0.05). E) Mitochondria were isolated from BMDMs, and ACSL1 abundance was determined by western blot with and without LPS treatment. The non-specific (ns) band serves as a loading control.